TW553745B - Microencapsulated oral vaccine - Google Patents

Abstract

The invention provides a process for producing oral microencapsulated or microsphere vaccine, comprising mixing an antigen suspension or solution with a water-soluble enteric coating and an excipient into a mixture, and co-spray drying the mixture to encapsulate the antigen and forming a microsphere.

Description

553745 ⑴

(Explanation of the invention should state: the technical field, the prior art, the content, the embodiments, and the drawings of the invention are briefly explained) The present invention proposes a vaccine process capable of producing microencapsules or microspheres, Enteric-soluble polymers (propionate-formate-propionate copolymers or modified formyl cellulose derivatives or modified acetylated cellulose derivatives) and excipients are dissolved in antigen suspension or aqueous solution with water Stir to form a mixed solution, and then cover and round the mixed solution by co-spray drying.

A vaccine is an antigenic biological preparation that can be applied to humans and animals. After the vaccine is administered, it can defame the production of antibodies and achieve the effect of resisting the invasion of pathogens. It is an important medicine for preventing human or animal infectious diseases. Generally, the vaccine products on the market are mainly in the form of injections. After inoculation in humans or animals, they can induce antibodies to produce immunity to specific diseases, which is a kind of preventive injection of artificial self-passive immunity. The present invention proposes another type of vaccine, which is designed to prevent diseases through oral administration.

We apply enteric polymer encapsulation technology to the antigen, which contains enteric preparations, which can pass through the stomach smoothly without being destroyed or broken down after oral administration. When it reaches the small intestine, the antigen is released immediately. Defaming the mucosa on the small intestine (such as Peyer's patch) to produce immunoglobulin has become the most effective first line of defense to prevent infection. In recent years, many reports have suggested that oral administration will be the safest, most convenient and economical immune route. Mucosal tissues can not only generate local immunity, but also transfer immune capacity to other mucosal tissues through special but not fully understood channels. Oral vaccines have the following advantages: (1) The immunization method is simple and safe. (2) The effect of secret antibody is better. (3) Compare to be patient or -6- (2)

Accepted by the owner. (4) Low cost. Making perfect oral vaccines and successfully delivering antigens to Peyer ’s patches via the gastrointestinal tract to defame the immune response has always been an important issue in immune control. The polymer coating material is used to contain small particles of the drug to form a micro-capsule container. This preparation is called a microcapsule or microsphere. Its particle size ranges from nm to several mm. The thickness, hardness, or dissolution characteristics of the membrane can be freely adjusted, so the appropriate amount of drug core or antigen can be effectively released at the appropriate time and where necessary. An ideal enteric coating material must have the following properties: (1) can resist gastric juice (2) the intestinal juice is sensitive and penetrable to it (3) has good compatibility with most pharmaceutical additives and the drug itself (Compatibility) (4) good stability (5) can form a continuous film (6) non-toxic (7) low cost (8) can be applied without special equipment. Commonly used as coating materials for enteric formulations include: cellulose acetate phthalate (CAP), methyl methacrylate methacrylic acid copolymer (trade names Eudragit L and Eudragits), hydroxy propyl methyl cellulose phthalate (HPMCP), cellulose modified derivatives (such as AQUOAT), polyvinyl acetate phthalate (PVAP), etc., are synthetic or semi-synthetic polymer substances that will only dissolve at a certain pH. These high-molecular substances do not dissolve under acidic conditions, but gradually dissolve in neutral or alkaline environments; in the end, the structure of the enteric preparations disintegrates and dissolves, so that the contents of the drug are completely released. The coating materials of formazan cellulose modified derivatives or acetylated cellulose modified derivatives (such as Aquacoal ECD) are time-dependent, and the disintegration time of the microcapsules can be prolonged according to the increase of the addition ratio. It is not affected by pH. However, the present invention uses a pH-dependent coating material (3) (3) 553,745, and uses materials such as HPMCP or AQ0A Ding and Aquac〇ai, in combination, the prepared microcapsules can protect the antigen from gastric acid damage, and When Tian reached the small intestine, the antigen was released due to rapid disintegration of pH. The traditional mouth spray drying film coating process is to inject the powder containing the antigen formula: the drug core is injected into the granulation tank in an appropriate amount, and the rolling of the granulation disc and the contact with the coating material sprayed and sprayed are passed through The hot air blowing and drying process can finally wrap the rolling antigen drug core with an enteric film coating. = This will complete the microencapsulated vaccine of the present invention. In the present invention, the easier method is used to homogenize the antigen with the aqueous solution of the enteric coating material and the excipient, and then directly co-spray drying simultaneously with the antigen coating and rounding. The present invention may use talc, glycerol or PEG as an excipient. The use of this simple synchronous spray drying technology to produce microcapsules has the following advantages: (1) sprayers are universal and readily available, (2) the manufacturing process is simple and low cost, (3) the output is large, and (4) a certain density can be continuously produced (5) The particle size is small and uniform. (5) The dry medium is hot air (37 < ^ ~ i4〇t), but the contact time between the particles and the hot gas is very short, and the latent heat of evaporation is absorbed by water. heat) and gasification, the particles themselves can still keep low temperature, suitable for drying of heat-resistant products (such as protein). Simultaneous spray drying procedures are simple, but still need a spray dryer and its main accessories, including: (1) a heater to heat the air to the required drying temperature, (2) an atomizer (atomizer)-the raw material or The coating material is sprayed into mist-like droplets. (3) Drying chamber—makes the wet droplets in full contact with the hot air. The water in the droplets quickly evaporates to become a dry powder. (4) Cyclone separation (4) 553745 (5) Extraction and exhaust (Cyclone separator)-recovery of product gas. Wide: The microcapsules of sexual vaccine can be directly mixed in the feed or mixed with organic acids .: The acid can be fed to the pigs overnight, and can be passed through the stomach without being destroyed and broken down. When it reaches the small intestine Antigen and intestinal enzyme inhibitor are released immediately: the inhibitor can inhibit the activity of intestinal enzymes, protect the released antigen so that it can reach the mucosal tissues in the small intestine, induce Peyer, spatens to produce an immune response, and prevent infection in pigs The first line of defense for mycoplasma pneumonia to reduce the loss of pig farmers is the safest, most convenient and economical route to immunization. With regard to the preparation of the microcapsules, the following examples are specifically explained, and are not limited to these examples. The picture uses water and gluten-free intestinal polymer methyl propionate mesylate · propionic acid copolymer as the microcapsule formula. After the microcapsule manufacturing is completed according to the formula in Table 1 and the synchronous spray drying method, the protein is dissolved by the dissolution test. Discussion on the release rate. Figure 2. Serum antibody results obtained by oral microcapsule mice after immunization. Figure 3. Antibody results obtained by oral microcapsule mice after immunization with intestinal wash. Figure 4. Antibody results obtained by oral microcapsule mice after immunization with lung wash. Water-soluble powdered enteric amidine molecule B brewed cellulose modified derivative as a microcapsule formula for protein release in different acid-base buffer solutions Production of Mycopiasma hyopneumoniae Oral Microcapsule Vaccine 553745 (5) This method is based on the formula shown in Table 1. The mycoplasma bacterial solution (10 ~ 82%), Water (52 ~ 0%) and glycerin (1 ~ 2%) are mixed. 'Add talc (0' ~ 8%) as a solidifying agent and fully disperse it. 'Then slowly add methyl propionate-propionate-propyl The intestine of the acetic acid copolymer is provided with a coating film (15% ~ 59%) of the coating material, and the liquid drips off. With a mouth mist dryer, it is blown with hot air at 37 ~ 14 ° C and 10,000 ~ 40,000 rpm. Microcapsules were prepared by simultaneous spray drying. After the finished products are collected by the cyclone, they can be stored in 4. Table 1. Examples of microcapsule preparation formulas (Εχ: Τ1Ε15, talc powder 1%, Eudragit 15%) treated mold mold glycerol hydrotalcite Eudragit Τ1Ε15 75ml 2ml 13 0ml 2.5g 37.5ml Τ1Ε30 75ml 2ml 92.5 ml 2.5g 75ml Τ3Ε15 7 5 ml 2ml 12 5ml 7.5g 37.5ml Τ3Ε30 7 5 ml 2ml 8 7.5ml 7.5g 7 5 ml Τ8Ε15 75ml 2ml 112.5ml 20g 37.5ml Τ8Ε30 75ml 2ml 7 5ml 20g 75ml

After the mold capsule liquid was manufactured according to the formula in Table 1 and the synchronous spray drying method was completed. The dissolution test was used to investigate the protein release rate. Figure 1 shows the protein release rate of each formula in pH7 neutral buffer (simulated intestinal fluid). It was found that the T1E15 and T1E30 formulas have the ability to release proteins quickly. Example 2: Immune response of enteric-coated microcapsule T1E15 in BALB / c mice model test. White mice BALB / c purchased from Taiwan National University of Animal Science, six to eight weeks old, were housed in standard animal rooms with air-conditioning equipment. , And supply rat feed (laboratory rodent diet # 5001) and clean drinking water. Animal test sample formula T 1 E 1 5 According to the relative amount of microcapsule-coated bacterial solution, samples of different antigen doses were prepared, and the samples were suspended in a 0.2% acetic acid aqueous solution. They were infused into the gastric sac and tested in 6 mice per experimental group. Gavage was performed every 10 days, and blood was drawn by eyeball extraction in the second week after three gavages, and autopsy of rats and sampling of lung and intestinal fluid were performed. The traditional injection vaccine was used as the positive control group. Similarly, it was injected once every ten days with 0.2 ml (equivalent to 0.114 ml of bacterial solution) each time. After three consecutive injections, samples were taken two weeks later. When collecting lung or intestinal lavage fluid, in order to prevent humoral enzymes from degrading antibody proteins, we use solution III (100 mg / ml soybean trypsin inhibitor, 50 mM EDTA, ImM PMSF, 0.51¼ gelatin, 0.05% NaN3 in PBS solution) as Rinse fluid, wash out lung or intestinal fluid immediately and store at -20 ° C until use. In terms of antibody detection, direct ELISA can be used for immunoquantitative analysis. The ELISA method is briefly described as follows: first, the amount of mycelial protein of 100 mg to the well is used as the first coating of the ELISA plate. When detecting antibodies, use appropriate amount of sample dilution solution in the well, and repeat six times for each sample. After a binding reaction at 37 ° C for one hour, then use goat anti-mouse IgG or IgA covalent AP (alkaline phosphatase conjugated) antibody. The binding reaction is performed and the color is reacted with p-nitrophenyl phosphate. The absorbance is detected at a wavelength of 405. Each ELISAplate needs a serial dilution of anti-mouse IgG, IgA antibody standard solution set. The reference calibration curve obtained from the reading can be used to convert the relative antibody concentration of the test sample. As shown in Figures 2, 3, and 4, mice received M.h · microcapsule oral vaccines, which were divided into three groups, T1E15 was the general oral group, Negative was fed the antigen-free microcapsule oral group, and Positive was the injection group. Gavage was taken every ten days for three consecutive gavages, and another two weeks later, blood was collected from the eye socket and IgG antibodies were detected. 553745

⑺ Injection M. h. Epidemic field was used as a positive control group, and its anti-M · h · IgG was obviously slandered. The concentration of the anti-M · h · IgG was about 20 ～ 40 ng / mi. In contrast, only about 12 ng / ml of IgG immune activity was produced in oral epidemic fields. Comparing the relationship between the dose and potency, it can be seen that the oral vaccine defies the IgG immune activity, but its effect is not as good as the injection. The immune activity of anti-M · h · IgA in intestinal and lung washes was detected. Although the concentration of IgA was much lower than that of IgG, we found that intestinal and lung washes after immunization in mice with microcapsule vaccine Significant IgA activity was detected in all of them. In the injection group, only a small amount of IgA activity was detected. In addition, statistical analysis was performed between the groups. In terms of serum, p < 001, showing a very significant difference (Figure 2). Intestinal washes showed significant differences between T1Ei5 and positive and Negative, and significant differences between T1E15 and positive (Figure 3). There was a significant difference between the groups in terms of lung lavage (Figure 4). According to the results of animal experiments in mice, an appropriate amount of M · h microcapsule vaccine can induce an immune response in mice by oral administration. This type of immune response is different from the general injection vaccine and should be local. Sexual immunity (local immunity). The use of the pan-mucosal immune system to prevent upper respiratory tract infections in pigs is another way to prevent mycoplasma suis, and its effectiveness is better than that of general injection vaccine injections, which needs to be evaluated on the spot. Only Example 3: Manufacturing modified mycoplasma hyopneumoniae oral microcapsule vaccine microcapsules with cellulose-derived derivatives as coating materials Enteric microcapsules can also be formulated. The mold pulp fungus solution (10 ~ 82%), hydroxy propyl methyl -12- 553745 ⑻ cellulosephthalate (HPMCP) (2 ~ 0%) and aqueous dispersion of cellulose derivatives (aqueous dispersion; 5 ~ 20%) After mixing and stirring well, the microcapsules were prepared by synchronous spray drying method under hot air blowing at 37 ~ 140 ° C and rotating speed of 10000 ~ 40,000 rpm. After the finished product is collected by the cyclone, it is stored at 4 ° C.

In order to understand such formulations, such as whether AQ6 is an enteric microcapsule, dissolution tests were performed in different pH buffers (ρΗ2, ρΗ3, ρΗ3 · 7, ρΗ4 · 5, ρΗ5 · 2, pH7). During the test period, approximately 1.5ml was sampled. After centrifugation at 15,000g, the supernatant was immediately stored in a freezer at -20 ° C. The quantified sample solution was diluted in PBS buffer, and protein was quantified using coomassie protein assay reagent. Figure 5 shows the protein release of different formulas in different acid-base buffer solutions. The main purpose is to discuss the protein release of modified derivatives of cellulose in acidic and neutral aqueous solutions. It is known from the results that the AQ6 formula has enteric properties. They do not dissolve protein in the acidic aqueous solution of ρΗ2-3, and the protein can be released at ρΗ5 · 2-7. In a neutral aqueous solution (ρΗ7) at 37 ° C, more than 90% of the protein is released after stirring for one hour, and a total of 97% is released in three hours. -13-

Claims (1)

553745 drying) for coating and rounding. 4. According to the process described in item 3 of the ifly * 1 range, HPMCP or aq_ ^ t modified cellulose derivatives can be added. 5. The process as described in item 4 of the scope of application, which can be used to prepare microcapsule vaccinia vaccines containing porcine pneumonia emblem bacterial culture antigen. 6. The process according to item 1 of the scope of patent application, wherein the prepared vaccine is an oral vaccine for animals or humans. 7. The process as described in item 3 of the scope of patent application, wherein the vaccine produced is an oral vaccine for animals or humans.